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Genetic Markers and Danger Signals in Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis
Allergology International. 2010;59:325-332 DOI: 10.2332! allergolint.10-RAI-0261
REVIEW ARTICLE
Genetic Markers and Danger Signals in Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis Wen-Hung Chung1 and Shuen-Iu Hung2 ABSTRACT Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse reactions, which could be induced by a variety of drugs. It was proposed that human leukocyte antigen (HLA)-restricted presentation of antigens (drugs or their metabolites) to T lymphocytes initiates the immune reactions of SJS! TEN. However, the genetic susceptibility and the exact pathogenesis were not clear until the recent studies. We first identified that HLA-B*1502 is strongly associated with carbamazepine (CBZ)-induced SJS!TEN and HLA-B*5801 with allopurinol-SJS!TEN in Han Chinese. The same associations had been validated across different human populations. For the downstream danger signals, Fas-Fas ligand (FasL) and perforin!granzyme B had been advocated as cytotoxic mediators for keratinocyte death in SJS!TEN. However, expression levels of these cytotoxic proteins from the skin lesions were too low to explain the distinct and extensive epidermal necrosis. Our recent study identified that the granulysin, a cytotoxic protein released from cytotoxic T cells or natural killer (NK) cells, is a key mediator for disseminated keratinocyte death in SJS!TEN. This article aims to provide an overview of both of the genomic and immunologic perspectives of SJS! TEN. These studies give us a better understanding of the immune mechanisms, biomarkers for disease prevention and early diagnosis, as well as providing the therapeutic targets for the treatments of SJS! TEN.
KEY WORDS drug hypersensitivity, genetic polymorphism, NK cells, Stevens-Johnson syndrome, toxic epidermal necrolysis
INTRODUCTION
ally “turns on” the extensive apoptosis in keratinocytes.1 In this article, we review the genomic and immunologic perspectives of SJS! TEN.
Drug hypersensitivity is a major clinical problem. Among the many types of drug hypersensitivity, SJS and TEN are the most serious and life-threatening adverse reactions. According to the clinical presentation and immunohistochemistry data, SJS and TEN have been considered as an immune disorder, involving both the adaptive and innate immune reactions. Applying techniques of pharmacogenomics and molecular biology in recent studies further revealed that the genetic disposition as well as immune mediators are important for the development of SJS and TEN. Although Fas-FasL interaction was previously considered to be the main effector in triggering apoptosis of keratinocytes, recent evidence suggested that granulysin, a cytotoxic protein produced by cytotoxic T lymphocytes (CTLs) and NK cells, is the one actu-
SJS! TEN are life-threatening severe cutaneous adverse reactions, presenting 10-40% mortality rate. SJS and TEN are classified as the same disease with different spectrums of severity according to the magnitude of epidermal detachment.2 Early symptoms of the abrupt onset of SJS usually start with fever, sore throat, and malaise, following by rapidly developing blistering exanthema of macules and target-like lesions accompanied mucosal involvement with less than 10% of skin detachment.2 TEN has similar clinical presentations with a more extensive separation of large sheets of epidermis from the dermis (greater than 30%) and a higher mortality rate (30-40%).3 The
1Department of Dermatology, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University and 2Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan. Correspondence: Wen-Hung Chung, Department of Dermatology,
Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, 199 Tung-Hwa North Road, Taipei 105, Taiwan. Email: [email protected] Received 31 August 2010. !2010 Japanese Society of Allergology
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CLINICAL MANIFESTATIONS OF SJS! TEN
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Chung WH et al. skin lesions usually begin on the trunk and spreads proximally in both of the SJS and TEN patients.2,3 Although having a low incidence, complications and its sequelae of SJS! TEN can results in death or disability in previously healthy people.4 Patients experiencing SJS! TEN are affected irreversible mucosal damage and the most serious ocular sequelae. Synechiae, corneal ulcers, symblepharon, and xerophthalmia are frequent ocular complications during the progression of SJS! TEN,5 and photophobia and xerophthalmia are commonly developed eye sequelae affected SJS! TEN survivals lifelong.6 Other internal organ involvements at mucosal surfaces are also common in SJS! TEN. With the increasing severity, abnormalities in respiratory tract,7 gastrointestior kidney are occasionally renal tract,8 liver, and! ported.9 The histopathology findings show that the significant feature of large portion epidermis separation from dermis is induced by massive keratinocyte apoptosis in SJS and TEN.10 Although Fas-FasL interaction was previously considered to be the main effector in triggering apoptosis of keratinocytes, evidence suggests that the granulysin1 is the one actually “turns on” apoptosis of keratinocyte.11 The main purpose of this review is to elucidate the pathomechanism of SJS and TEN in genetic and immunologic point of views.
GENETIC MARKERS RELATED TO SJS! TEN Genetic association between HLA alleles and SJS! TEN was found from several reports, and the ethnicity specific feature is emphasized these years. A significantly strong correlation was first found in Han TEN patients Chinese in 2004.12 CBZ-induced SJS! carried 100% of HLA-B*1502 allele, and only 3% HLAB*1502 carriers tolerated CBZ.12 According to its absence in Caucasians13 and Japanese,14 HLA-B*1502 allele seems uniquely limited in Han Chinese ancestral Asians and might be an explanation for the extremely high risk of CBZ-induced SJS! TEN in Southeast Asians comparing to Caucasians and Japanese. Later studies conducted in the South-East countries, including Hong Kong, Malaysia, India, Singapore, Thailand, Vietnam, Indonesia, and Philippines, were further confirming its high prevalence (2.3% to 8.4%) of this ethnic specific allele.15 Because of the ethnic specialty in HLA-B*1502, CBZ and some similar structural anticonvulsants were fully studied. One hundred percent CBZ-induced SJS was HLA-B*1502 in Thailand people.16 Malay and Chinese carried 15.7% and 5.7% of HLA-B*1502, respectively, and had 75% incidence of HLA-B*1502 in CBZ-SJS or TEN.17 A lower prevalence (2.5%)18 and 75% incidence of HLA-B*1502 in CBZ-induced SJS were reported in Indians.19 Other than HLA-B*1502, HLA-B*5901 has been suggested as a candidate marker in CBZ-induced SJS in Japanese with 15.16 relative risk20; however, due to
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its small sample size (10 patients), further investigation is needed to validate this finding. In addition, HLA-A*0206, was proposed as a marker in SJS! TEN according to the ocular complications in Japanese.21 HLA-B*5801 allele is another genetic marker found to have an association with a commonly prescribed drug for decreasing uric acid level in hyperuricemia―allopurinol. In Taiwanese, it was 100% presented in patients with allopurinol-induced severe cutaneous adverse reactions, but only 15% in allopurinol tolerant patients.22 The same strong association was confirmed in a Thai population.23 However, findings from Japanese population remain to clarify. A moderate association (4 out of 10) in a Japanese population14 was reported. Opposite to this finding, Kano et al. suggested that HLA-B*4801 but not B*5801 was associated with CBZ-induced drug rash with eosinophilia and systemic symptoms (DRESS).24 In addition, the ethnic limitation also presents in HLA-B* 5801 on its low allele frequency in Caucasians.25 Moreover, an antiretroviral drug, abacavir, can cause drug hypersensitivity and was linked to HLA-B* 5701 allele. The combination of HLA-B*5701, HLADR7, and HLA-DQ3 once reported having a 100% predictability of the abacavir-induced hypersensitivity.26 Further study demonstrated that the prevalence of HLA-B*5701 was high in Caucasians and suggested that prior screening of the carriage of HLA-B*5701 could effectively reduce the abacavir hypersensitivity.27 In contrast to the high prevalence of HLA-B* 5701 and incidence of abacavir hypersensitivity, none of the patients with Asian ancestry in Korea carried HLA-B*5701.28
DRUG-SPECIFIC, HLA-DEPENDENT, T-CELLMEDIATED IMMUNITY IN SJS! TEN The discoveries of the involvement of HLAdependent presentation of an offending drug or its metabolites for T-cell activation29,30 demolished the “hapten hypothesis”31 in severe drug hypersensitivity. The proposed pharmoco-immune (p-i) concept32 is the mainstream explanation for the drug-induced delay cutaneous adverse reactions, suggesting the direct and non-covalent binding between a drug and Tcell receptors (TCR) with HLA molecules takes the responsibility for the drug-induced immunity (Fig. 1). The pathogenesis of the induction of cytotoxic responses in SJS! TEN is generated by the recognition of offending drugs to HLA class I molecule initiated T-cell activation which results a clonal expansion of CD8+ cytotoxic T-cells in skin.33 Our findings, the strong associations between HLA-B*1502 and CBZ12 as well as the HLA-B*5801 and allopurinol,22 support that drug-induced SJS! TEN is a HLA-restricted immunity.12 Furthermore, our later results verified 5 peptides showing high affinities for HLA-B*1502, providing CBZ recognition of HLA, locating at antigen presenting cells.34,35
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Genetics and Signals in SJS! TEN
CBZ
HLAB*1502
Antigen(drug) CD8 APC (Keratinocyte)
MHC class I
Keratinocyte Apoptosis
CD8+ Cytotoxic T-cell TCR
Granulysin
Fi g.1 A modelofker at i noc y t eapopt os i si nduc edbyt hei mmune synapseofdr ugHLATCR i nt er ac t i oni nSJ S/ TEN.Asi l l us t r at ed,t he i mmuner esponsemaybet r i gger edbyt hebi ndi ngofanant i geni c dr ug( e. g.car bamazepi ne[ CBZ] ) ,t oas pec i f i cHLAal l el e( e. g.HLAB* 1502)onaker at i noc y t e,whi c har et hemai nant i genpr es ent i ng cel l s( APC)i nSJS/ TEN.Then,s pec i f i cTc el lr ec ept or s( TCR)oft he CD8+ cyt ot oxi cT l ymphoc y t es ( CTLs )r ec ogni z et he dr ugHLA compl ex.Upont heact i v at i on,CTLsorNKTc el l spr oduc ec y t ok i nes andchemoki nes,aswel last hec y t ot ox i cpr ot ei ns ,par t i c ul ar l y ,s ec r et or ygr anul ysi nandl eadt oex t ens i v ek er at i noc y t eapopt os i s .
INVOLVEMENT OF NATURAL KILLER CELLS IN SJS! TEN In addition to CTLs, NK cells also involves in SJS! TEN. The blister cells in skin lesions of SJS! TENaffected patients were mainly CTLs and NK cells36; moreover, HLA class I-binding proteins have activating (KAR, killer activating receptor) or inhibitory (KIR, killer inhibitory receptor) cytolytic functions for regulating NK cells.37 Nassif A. et al.29 further demonstrated an observation of the infiltration of CTLs and NK cells in skin lesions in TEN patients. Our recent study also demonstrated that granulysin, secreted from CTLs and NK cells, is a key effector responsible for keratinocytes death in SJS! TEN.1
DANGER SIGNALS INDUCING EXTENSIVE KERATINOCYTE APOPTOSIS IN SJS! TEN Upon proposed immunopathogenesis of SJS! TEN, the general agreed central hypothesis is the T-cellmediated massive apoptosis in keratinocytes. Until recently, there are three reported pathways advocated as basic effectors: Fas-FasL interaction, perforin! granzyme B, and the granulysin.
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Fas-FasL-INDUCED APOPTOSIS Although Fas-FasL-induced apoptosis in keratinocytes is one of the most thoroughly studied immune mechanism in SJS! TEN, inconsistent findings of Fas, FasL, and soluble FasL (sFasL) questioned the original hypothesis from Viard et al.11 Fas was discovered to cause cell death upon binding with its ligand in SJS! TEN. Viard et al.11 suggested the activated Fas servers as a death receptor in triggering apoptosis of keratinocytes in SJS! TEN. The cytoplasmic death domain of Fas undergoes conformational changes upon recognition of FasL. The Fas-FasL then recruits a Fas-associated death domain protein (FADD) which has a affinity to bind to both of the Fas death domain and procaspase 8. Once the procaspase 8 is recruited by FADD, the multiple copies of procaspase 8 are brought together and autoactivate themselves to caspase 8 which triggers the caspase cascade for intracellular DNA degradation.38 One study concluded the same result as Viard et al. by incubating cell-free supernatants of blister fluid containing sFasL secreted from keratinocytes and not resulting in apoptosis in keratinocytes.39 Viard et al. also showed that FasL presented on the
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Chung WH et al. cell surface of keratinocytes in TEN patients and sFasL was found to have high levels in the serum, but not in patients with maculopapular drug reaction or normal persons.11 Metalloproteinases (MPs) present at cell surface in many tissue types, including keratinocytes40 and cleave FasL into sFasL at its TNFhomologous portion.41,42 The high sFasL serum level observed in TEN patients might cause by the MP cleavage reaction. A study also consistently found an apparent elevation of sFasL within 2 days after the onset of skin damage in a TEN patient.43 Testing the serological sFasL for a short period at the beginning of onset may help to define the progress of SJS! TEN. Controversial interpretations disagreed with either the source of FasL or its role in apoptosis effector. Studies demonstrated that FasL is not located at the extracellular membrane surface of keratinocytes, but rather transporting to the cell surface upon suffering keratinocyte damage.40,44 Abe et al.45 found that the apoptosis of cultured keratinocytes was induced by adding high levels of sFasL containing sera isolated from a SJS! TEN patient, and was blocked by addition of anti-FasL monoclonal antibody. They also showed the peripheral blood mononuclear cells (PBMCs) of TEN patients were the sites produced high levels of sFasL.45 Since Amato et al.46 first reported the treatment of intravenous immunoglobulins (IVIG) in a SJS patient, many studies subsequently applied IVIG to treat the SJS and TEN patients. IVIG therapy is a treatment based on the hypothesis of Fas-FasL interaction, by blocking FasL binding to the Fas receptor interferes with the downstream signaling for triggering apoptosis of the keratinocytes.11 Although evidence seemed to show the effectiveness of IVIG in in vitro studies,11,47 benefits were inconsistently seen in neither the mortality nor the progression of skin detachment,48-52 which yield the controversial role for the IVIG treatment in SJS! TEN.
PERFORIN! GRANZYME B APOPTIC PATHWAY High concentrations of granzyme B was found in TEN blister fluid.53 Nassif A. et al.54 presented a contradictory hypothesis to the Fas-FasL interaction by showing that the cytotoxicity from the blister fluid mononuyclear cells in TEN could be blocked by inhibitors of perforin! granyzyme B rather then blocked by an anti-Fas monoclonal antibody. Perforin and granzyme B are stored in secretory granules of activated CTLs and NK cells.55 Perforin binds and punches a channel in the membrane of target cells for entering of the granzyme B to activate the caspase cascade and its following apoptotic pathways.56,57
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OTHER CYTOKINES AND SIGNALS INVOLVED IN THE PATHOGENESIS OF SJS! TEN In addition to Fas-FasL or perforin! granzyme B pathways, some cytokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ,39 and interleukin (IL)-10,39 were also found to be up-regulated in SJS! TEN. Blister cells of SJS! TEN were reported to secret IFN-γ and stimulate keratinocytes to express TNF-α, FasL, and IL-10 which were present in higher concentrations in the blister fluids as a defense mechanism against CTLs.39 TNF-α has been reported abundantly presenting in the keratinocytes of epidermis,58,59 blister fluid mononuclear cells and macroTEN. TNF-α has phages53,58,59 and PBMCs60 in SJS! been suggested having an up-regulating function in Fas and FasL60 and via the activation of TNF-receptor 1 (TNF-R1), initiating the downstream FADD and caspases.61,62 Apoptosis activation in TEN patients had been implied to be induced by TNF.10 In addition, little evidence showed that TNF-α also can activate TNF-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-Rs), which may result in the activation of FADD apoptotic pathway.63 Interestingly, contradictory to TNF-α apoptosis inducing role, its binding to TNF-R1 can up-regulate NF-κB64 and associate with an anti-apoptotic in keratinocytes.65 Anti-TNF therapy once was thought to be a possible potential treatment for TEN patients; however, no beneficial effect was concluded from anti-TNF by thalidomide in TEN.66 There was one study that supported both of the Fas-FasL and perforin! granzyme B pathways in keratinocytes apoptosis.53 This study found an increasing pattern of TNF-α, perforin, granzyme B, and FasL accompanied with the severity, ranging from the mildest maculopapular rashes to the severest TEN, and suggested that the perforin! granzyme B played as the major effectors and Fas-FasL interaction was probably related to the severer adverse drug reactions.
GRANULYSIN IS THE MAJOR FACTOR FOR KERATINOCYTE APOPTOSIS IN SJS! TEN Recently, we found secretory granulysin servers as a major cytotoxic molecule responsible for widespread keratinocyte necrosis in SJS! TEN,1 instead of those previously reported sFasL, granzyme B, or perforin. We performed global gene expression profiling of the blister cells and found that granulysin RNA was the most significant cytotoxic molecule expressed. Granulysin protein concentrations in the SJS! TEN blister fluids were two to four orders of magnitude higher than perforin, granzyme B or sFasL concentrations, and depleting granulysin reduced the cytotoxicity (Fig. 2). Westernblot analysis showed that granulysin in the fluids was predominantly the 15-
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Genetics and Signals in SJS! TEN
Keratinocyte
CLT
Epidermis
NK/NKT Apoptotic keratinocyte
Blister
sFasL Perforin Grazyme B
Blood vessel
Fibroblast
Dermis
Granulysin Caspase
Fi g.2 Pat hogenesi sofepi der malnecr osi sanddi s s emi nat edk er at i noc y t eapopt os i si nSJ S/ TEN.Duet ot he si gnal sofi mmunesynapse,t heCTLsandNK/ NKTc el l si mmi gr at et ot heepi der mi sofs k i n.CTLs ,andNK/ NKT cel l spr oduceal ar geamountofi mmunemedi at or s( e. g. ,s ol ubl eFas L[ s Fas L] ,per f or i n,gr az y meB,andgr anul ysi n)i nt ot heext r acel l ul arspace.I npar t i cul ar ,s ec r et or ygr anul y s i n,ex pr es s edatav er yhi ghl ev eli nt hes k i nl esi ons,i samai nweaponofCTLs,NK,andNKTc el l s ,at t ac k sk er at i noc y t esandr es ul t si nex t ens i v eepi der mal necr osi sandbl i st erf or mat i on.Bycompar i son,gr anz y meB/ per f or i nandFas / Fas Lar epr oduc edv i agr anul eex ocyt osi suponcel l cel l cont act ,andpr esenti nl owerc onc ent r at i onst hangr anul y s i ni nt hei nf l ammat or yl es i ons .Af t erencount er i ng t he at t acksoft hese cyt ot ox i cpr ot ei ns ,k er at i noc y t esar e damaged and t hen t he c as pas e si gnal i ngpat hwayt ur nson,l eadi ngt oapopt os i spr ogr es s .
kDa secretory form. In vitro, purified 15-kDa granulysin exhibited significant cytotoxicity at the level presenting in the SJS! TEN blister fluids. However, sFasL, perforin, and granzyme B concentrations in the SJS! TEN blister fluid had minimum cytotoxicity. A further injection of granulysin into mouse skin resulted in a SJS-TEN-alike skin necrosis.1 Thus, our findings demonstrated that granulysin, not granzyme B, perforin or sFasL as previously implicated, is the key molecule responsible for the disseminated keratinocyte death in SJS! TEN. Following this line, Abe et al.45 further reported that an increasing serum level of granulysin could serve as an early diagnostic biomarker for SJS! TEN. Granulysin is a cationic cytolytic protein produced by CTLs, NK and NKT cells.67 The 15-kDa granulysin was proposed as a precursor of the 9-kDa form.68,69 In addition to having the cytotoxic effect, granulysin also was demonstrated its chemoattractant ability for T lymphocytes, monocytes and other inflammatory cells, and activation function in the expression of a number of cytokines, including RANTES! CCL5, MCP-1, MCP-3, MIP-1α! CCL3, IL-10, IL-1, IL-6 and
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IFN-α.70 However, much of SJS! TEN remains in mystery. How does taking a drug lead to secretion of granulysin? How does CD8+T! NK and NKT cells regulate the secretion of granulysin in SJS! TEN? The specific relationship among offending drugs, HLA alleles, and cytotoxic signals from CLTs! NK! NKT cells in SJS! TEN remains further investigation.
CONCLUSION Increasing data have revealed that the genetic predisposition and immune mediators play important roles in the drug hypersensitivity. As reviewed in this article, it is known that HLA alleles associated with SJS! TEN may be variable among different human population. These HLA alleles may be not only responsible for the genetic susceptibility, but also play a pathogenesis role, which may present the drugs! metabolites to CTLs for the initiation of the downstream danger signals in the disease. In addition to Fas-FasL, perforin! granzyme B biomarkers, the secretory 15 kDa granulysin, is now known to be the major weapon of CTLs! NK! NKT cells and leads to the ex-
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Chung WH et al. tensive epidermal necrolysis in SJS! TEN. Understanding the molecular mechanism of the interaction of HLA, offending drugs and TCR, as well as CTLs! NK cells activation, would facilitate the development of new approaches for the management of SJS! TEN. In conclusion, those transitional studies offer us a better understanding of the immune mechanisms, biomarkers for disease prevention and early diagnosis, as well as providing the therapeutic target for the treatment of SJS! TEN.
ACKNOWLEDGEMENTS This work was supported by grants from the National Science Council, Taiwan (NSC 98-2314-B-182A-027MY3, 98-2320-B-010-002-MY3), and grants from Chang-Gung Memorial Hospital (BMRPG-290011, CMRPG-290051).
REFERENCES 1. Chung WH, Hung SI, Yang JY et al. Granulysin is a key mediator for disseminated keratinocyte death in StevensJohnson syndrome and toxic epidermal necrolysis. Nat Med 2008;14:1343-50. 2. Roujeau JC. The spectrum of Stevens-Johnson syndrome and toxic epidermal necrolysis: a clinical classification. J Invest Dermatol 1994;102:28S-30. 3. Roujeau JC, Stern RS. Severe adverse cutaneous reactions to drugs. N Engl J Med 1994;331:1272-85. 4. Chan HL, Stern RS, Arndt KA et al. The incidence of erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis. A population-based study with particular reference to reactions caused by drugs among outpatients. Arch Dermatol 1990;126:43-7. 5. Di Pascuale MA, Espana EM, Liu DT et al. Correlation of corneal complications with eyelid cicatricial pathologies in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis syndrome. Ophthalmology 2005;112: 904-12. 6. Revuz J, Penso D, Roujeau JC et al. Toxic epidermal necrolysis. Clinical findings and prognosis factors in 87 patients. Arch Dermatol 1987;123:1160-5. 7. Lebargy F, Wolkenstein P, Gisselbrecht M et al. Pulmonary complications in toxic epidermal necrolysis: a prospective clinical study. Intensive Care Med 1997;23:123744. 8. Sugimoto Y, Mizutani H, Sato T, Kawamura N, Ohkouchi K, Shimizu M. Toxic epidermal necrolysis with severe gastrointestinal mucosal cell death: a patient who excreted long tubes of dead intestinal epithelium. J Dermatol 1998;25:533-8. 9. Ducic I, Shalom A, Rising W, Nagamoto K, Munster AM. Outcome of patients with toxic epidermal necrolysis syndrome revisited. Plast Reconstr Surg 2002;110:768-73. 10. Paul C, Wolkenstein P, Adle H et al. Apoptosis as a mechanism of keratinocyte death in toxic epidermal necrolysis. Br J Dermatol 1996;134:710-4. 11. Viard I, Wehrli P, Bullani R et al. Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. Science 1998;282:490-3. 12. Chung WH, Hung SI, Hong HS et al. Medical genetics: a marker for Stevens-Johnson syndrome. Nature 2004;428: 486. 13. Lonjou C, Thomas L, Borot N et al. A marker for Stevens-
330
Johnson syndrome...: ethnicity matters. Pharmacogenomics J 2006;6:265-8. 14. Kaniwa N, Saito Y, Aihara M et al. HLA-B locus in Japanese patients with anti-epileptics and allopurinol-related Stevens-Johnson syndrome and toxic epidermal necrolysis. Pharmacogenomics 2008;9:1617-22. 15. Allele Frequencies in Worldwide Populations. Available from http:! ! www.allelefrequencies.net! . 16. Locharernkul C, Loplumlert J, Limotai C et al. Carbamazepine and phenytoin induced Stevens-Johnson syndrome is associated with HLA-B*1502 allele in Thai population. Epilepsia 2008;49:2087-91. 17. Chang CC, Too CL, Murad S. Association of HLA-B*1502 with carbamazepine-induced toxic epidermal necrolysis and Stevens-Johnson Syndrome in Malaysian population. Proceeding in 7th Asian-Oceanian Epilepsy Congress, Xiamen. 2008. 18. Rajalingam R, Parham P, Mehra NK. HLA-A, -B, -Cw, -DQA1, -DQB1 and -DRB1 alleles and KIR alleles in a Hindu population from Punjab, India. Human Immunology 2004;65:958-60. 19. Mehta TY, Prajapati LM, Mittal B et al. Association of HLA-B*1502 allele and carbamazepine-induced StevensJohnson syndrome among Indians. Indian J Dermatol Venereol Leprol 2009;75:579-82. 20. Ikeda H, Takahashi Y, Yamazaki E et al. HLA class I markers in Japanese patients with carbamazepineinduced cutaneous adverse reactions. Epilepsia 2010;51: 297-300. 21. Ueta M, Tokunaga K, Sotozono C et al. HLA class I and II gene polymorphisms in Stevens-Johnson syndrome with ocular complications in Japanese. Mol Vis 2008;14:550-5. 22. Hung SI, Chung WH, Liou LB et al. HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol. Proc Natl Acad Sci U S A 2005; 102:4134-9. 23. Tassaneeyakul W, Jantararoungtong T, Chen P et al. Strong association between HLA-B*5801 and allopurinolinduced Stevens-Johnson syndrome and toxic epidermal necrolysis in a Thai population. Pharmacogenet Genomics 2009;19:704-9. 24. Kano Y, Hirahara K, Asano Y, Shiohara T. HLA-B allele associations with certain drugs are not confirmed in Japanese patients with severe cutaneous drug reactions. Acta Derm Venereol 2008;88:616-8. 25. Lonjou C, Borot N, Sekula P et al. A European study of HLA-B in Stevens-Johnson syndrome and toxic epidermal necrolysis related to five high-risk drugs. Pharmacogenet Genomics 2008;18:99-107. 26. Mallal S, Nolan D, Witt C et al. Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to HIV-1 reverse-transcriptase inhibitor abacavir. Lancet 2002;359:727-32. 27. Mallal S, Phillips E, Carosi G et al. HLA-B*5701 screening for hypersensitivity to abacavir. N Engl J Med 2008;358: 568-79. 28. Park WB, Choe PG, Song KH et al. Should HLA-B*5701 screening be performed in every ethnic group before starting abacavir? Clin Infect Dis 2009;48:365-7. 29. Nassif A, Bensussan A, Boumsell L et al. Toxic epidermal necrolysis: effector cells are drug-specific cytotoxic T cells. J Allergy Clin Immunol 2004;114:1209-15. 30. Schnyder B, Mauri-Hellweg D, Zanni M, Bettens F, Pichler WJ. Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alphabeta T cell clones. J Clin Invest 1997;100:136-41.
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31. Weltzien HU, Moulon C, Martin S, Padovan E, Hartmann
49. Schneck J, Fagot JP, Sekula P, Sassolas B, Roujeau JC,
U, Kohler J. T cell immune responses to haptens. Structural models for allergic and autoimmune reactions. Toxicology 1996;107:141-51. 32. Pichler WJ. Pharmacological interaction of drugs with antigen-specific immune receptors: the p-i concept. Curr Opin Allergy Clin Immunol 2002;2:301-5. 33. Naisbitt DJ, Britschgi M, Wong G et al. Hypersensitivity reactions to carbamazepine: characterization of the specificity, phenotype, and cytokine profile of drug-specific T cell clones. Mol Pharmacol 2003;63:732-41. 34. Yang CW, Hung SI, Juo CG et al. HLA-B*1502-bound peptides: implications for the pathogenesis of carbamazepineinduced Stevens-Johnson syndrome. J Allergy Clin Immunol 2007;120:870-7. 35. Barber LD, Percival L, Arnett KL, Gumperz JE, Chen L, Parham P. Polymorphism in the alpha 1 helix of the HLAB heavy chain can have an overriding influence on peptide-binding specificity. J Immunol 1997;158:1660-9. 36. David V, Bourge JF, Guglielmi P, Mathieu-Mahul D, Degos L, Bensussan A. Human T cell clones use a CD3associated surface antigen recognition structure to exhibit both NK-like and allogeneic cytotoxic reactivity. J Immunol 1987;138:2831-6. 37. Le Cleach L, Delaire S, Boumsell L et al. Blister fluid T lymphocytes during toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors. Clin Exp Immunol 2000;119: 225-30. 38. Janeway CA, Travers P, Walport M, Schlomchik M. Immunobiology: The Immune System in Health and Disease, 5th edn. New York: Garland Publishing, 2001. 39. Nassif A, Moslehi H, Le Gouvello S et al. Evaluation of the potential role of cytokines in toxic epidermal necrolysis. J Invest Dermatol 2004;123:850-5. 40. Viard-Leveugle I, Bullani RR, Meda P et al. Intracellular localization of keratinocyte Fas ligand explains lack of cytolytic activity under physiological conditions. J Biol Chem 2003;278:16183-8. 41. Tanaka M, Itai T, Adachi M, Nagata S. Downregulation of Fas ligand by shedding. Nat Med 1998;4:31-6. 42. Knox PG, Milner AE, Green NK, Eliopoulos AG, Young LS. Inhibition of metalloproteinase cleavage enhances the cytotoxicity of Fas ligand. J Immunol 2003;170:677-85. 43. Chang HY, Cooper ZA, Swetter SM, Marinkovich MP. Kinetics and specificity of fas ligand induction in toxic epidermal necrolysis. Arch Dermatol 2004;140:242-4. 44. Filipowicz E, Adegboyega P, Sanchez RL, Gatalica Z. Expression of CD95 (Fas) in sun-exposed human skin and cutaneous carcinomas. Cancer 2002;94:814-9. 45. Abe R, Shimizu T, Shibaki A, Nakamura H, Watanabe H, Shimizu H. Toxic epidermal necrolysis and StevensJohnson syndrome are induced by soluble Fas ligand. Am J Pathol 2003;162:1515-20. 46. Amato GM, Travia A, Ziino O. [The use of intravenous high-dose immunoglobulins (IGIV) in a case of StevensJohnson syndrome]. Pediatr Med Chir 1992;14:555-6 (in Italian). 47. Wehrli P, Viard I, Bullani R, Tschopp J, French LE. Death receptors in cutaneous biology and disease. J Invest Dermatol 2000;115:141-8. 48. Bachot N, Revuz J, Roujeau JC. Intravenous immunoglobulin treatment for Stevens-Johnson syndrome and toxic epidermal necrolysis: a prospective noncomparative study showing no benefit on mortality or progression. Arch Dermatol 2003;139:33-6.
Mockenhaupt M. Effects of treatments on the mortality of Stevens-Johnson syndrome and toxic epidermal necrolysis: A retrospective study on patients included in the prospective EuroSCAR Study. J Am Acad Dermatol 2008;58: 33-40. 50. Valeyrie-Allanore L, Wolkenstein P, Brochard L et al. Open trial of ciclosporin treatment for Stevens-Johnson syndrome and toxic epidermal necrolysis. Br J Dermatol. Epub 2010 May 25. 51. Wolf R, Davidovici B. Severe cutaneous adverse drug reactions: who should treat, where and how?: Facts and controversies. Clin Dermatol 2010;28:344-8. 52. Mittmann N, Chan B, Knowles S, Cosentino L, Shear N. Intravenous immunoglobulin use in patients with toxic epidermal necrolysis and Stevens-Johnson syndrome. Am J Clin Dermatol 2006;7:359-68. 53. Posadas SJ, Padial A, Torres MJ et al. Delayed reactions to drugs show levels of perforin, granzyme B, and Fas-L to be related to disease severity. J Allergy Clin Immunol 2002;109:155-61. 54. Nassif A, Bensussan A, Dorothee G et al. Drug specific cytotoxic T-cells in the skin lesions of a patient with toxic epidermal necrolysis. J Invest Dermatol 2002;118:728-33. 55. Tschopp J, Nabholz M. Perforin-mediated target cell lysis by cytolytic T lymphocytes. Annu Rev Immunol 1990;8: 279-302. 56. Lowin B, Peitsch MC, Tschopp J. Perforin and granzymes: crucial effector molecules in cytolytic T lymphocyte and natural killer cell-mediated cytotoxicity. Curr Top Microbiol Immunol 1995;198:1-24. 57. Darmon AJ, Nicholson DW, Bleackley RC. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature 1995;377:446-8. 58. Paquet P, Nikkels A, Arrese JE, Vanderkelen A, Pierard GE. Macrophages and tumor necrosis factor alpha in toxic epidermal necrolysis. Arch Dermatol 1994;130:6058. 59. Paquet P, Pierard GE. Erythema multiforme and toxic epidermal necrolysis: a comparative study. Am J Dermatopathol 1997;19:127-32. 60. Arnold R, Seifert M, Asadullah K, Volk HD. Crosstalk between keratinocytes and T lymphocytes via Fas! Fas ligand interaction: modulation by cytokines. J Immunol 1999;162:7140-7. 61. Zhuang L, Wang B, Shinder GA, Shivji GM, Mak TW, Sauder DN. TNF receptor p55 plays a pivotal role in murine keratinocyte apoptosis induced by ultraviolet B irradiation. J Immunol 1999;162:1440-7. 62. Ruckert R, Lindner G, Bulfone-Paus S, Paus R. High-dose proinflammatory cytokines induce apoptosis of hair bulb keratinocytes in vivo. Br J Dermatol 2000;143:1036-9. 63. Leverkus M, Neumann M, Mengling T et al. Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes. Cancer Res 2000;60:553-9. 64. Wang CY, Cusack JC Jr, Liu R, Baldwin AS Jr. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaB. Nat Med 1999;5:412-7. 65. Qin JZ, Chaturvedi V, Denning MF, Choubey D, Diaz MO, Nickoloff BJ. Role of NF-kappaB in the apoptoticresistant phenotype of keratinocytes. J Biol Chem 1999; 274:37957-64. 66. Wolkenstein P, Latarjet J, Roujeau JC et al. Randomised comparison of thalidomide versus placebo in toxic epider-
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mal necrolysis. Lancet 1998;352:1586-9.
67. Gamen S, Hanson DA, Kaspar A, Naval J, Krensky AM, Anel A. Granulysin-induced apoptosis. I. Involvement of at least two distinct pathways. J Immunol 1998;161:1758-64. 68. Stenger S, Hanson DA, Teitelbaum R et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998;282:121-5. 69. Hanson DA, Kaspar AA, Poulain FR, Krensky AM. Bio-
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synthesis of granulysin, a novel cytolytic molecule. Mol Immunol 1999;36:413-22. 70. Deng A, Chen S, Li Q, Lyu SC, Clayberger C, Krensky AM. Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator. J Immunol 2005; 174:5243-8.
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REVIEW ARTICLE
Genetic Markers and Danger Signals in Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis Wen-Hung Chung1 and Shuen-Iu Hung2 ABSTRACT Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening adverse reactions, which could be induced by a variety of drugs. It was proposed that human leukocyte antigen (HLA)-restricted presentation of antigens (drugs or their metabolites) to T lymphocytes initiates the immune reactions of SJS! TEN. However, the genetic susceptibility and the exact pathogenesis were not clear until the recent studies. We first identified that HLA-B*1502 is strongly associated with carbamazepine (CBZ)-induced SJS!TEN and HLA-B*5801 with allopurinol-SJS!TEN in Han Chinese. The same associations had been validated across different human populations. For the downstream danger signals, Fas-Fas ligand (FasL) and perforin!granzyme B had been advocated as cytotoxic mediators for keratinocyte death in SJS!TEN. However, expression levels of these cytotoxic proteins from the skin lesions were too low to explain the distinct and extensive epidermal necrosis. Our recent study identified that the granulysin, a cytotoxic protein released from cytotoxic T cells or natural killer (NK) cells, is a key mediator for disseminated keratinocyte death in SJS!TEN. This article aims to provide an overview of both of the genomic and immunologic perspectives of SJS! TEN. These studies give us a better understanding of the immune mechanisms, biomarkers for disease prevention and early diagnosis, as well as providing the therapeutic targets for the treatments of SJS! TEN.
KEY WORDS drug hypersensitivity, genetic polymorphism, NK cells, Stevens-Johnson syndrome, toxic epidermal necrolysis
INTRODUCTION
ally “turns on” the extensive apoptosis in keratinocytes.1 In this article, we review the genomic and immunologic perspectives of SJS! TEN.
Drug hypersensitivity is a major clinical problem. Among the many types of drug hypersensitivity, SJS and TEN are the most serious and life-threatening adverse reactions. According to the clinical presentation and immunohistochemistry data, SJS and TEN have been considered as an immune disorder, involving both the adaptive and innate immune reactions. Applying techniques of pharmacogenomics and molecular biology in recent studies further revealed that the genetic disposition as well as immune mediators are important for the development of SJS and TEN. Although Fas-FasL interaction was previously considered to be the main effector in triggering apoptosis of keratinocytes, recent evidence suggested that granulysin, a cytotoxic protein produced by cytotoxic T lymphocytes (CTLs) and NK cells, is the one actu-
SJS! TEN are life-threatening severe cutaneous adverse reactions, presenting 10-40% mortality rate. SJS and TEN are classified as the same disease with different spectrums of severity according to the magnitude of epidermal detachment.2 Early symptoms of the abrupt onset of SJS usually start with fever, sore throat, and malaise, following by rapidly developing blistering exanthema of macules and target-like lesions accompanied mucosal involvement with less than 10% of skin detachment.2 TEN has similar clinical presentations with a more extensive separation of large sheets of epidermis from the dermis (greater than 30%) and a higher mortality rate (30-40%).3 The
1Department of Dermatology, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University and 2Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan. Correspondence: Wen-Hung Chung, Department of Dermatology,
Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, 199 Tung-Hwa North Road, Taipei 105, Taiwan. Email: [email protected] Received 31 August 2010. !2010 Japanese Society of Allergology
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CLINICAL MANIFESTATIONS OF SJS! TEN
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Chung WH et al. skin lesions usually begin on the trunk and spreads proximally in both of the SJS and TEN patients.2,3 Although having a low incidence, complications and its sequelae of SJS! TEN can results in death or disability in previously healthy people.4 Patients experiencing SJS! TEN are affected irreversible mucosal damage and the most serious ocular sequelae. Synechiae, corneal ulcers, symblepharon, and xerophthalmia are frequent ocular complications during the progression of SJS! TEN,5 and photophobia and xerophthalmia are commonly developed eye sequelae affected SJS! TEN survivals lifelong.6 Other internal organ involvements at mucosal surfaces are also common in SJS! TEN. With the increasing severity, abnormalities in respiratory tract,7 gastrointestior kidney are occasionally renal tract,8 liver, and! ported.9 The histopathology findings show that the significant feature of large portion epidermis separation from dermis is induced by massive keratinocyte apoptosis in SJS and TEN.10 Although Fas-FasL interaction was previously considered to be the main effector in triggering apoptosis of keratinocytes, evidence suggests that the granulysin1 is the one actually “turns on” apoptosis of keratinocyte.11 The main purpose of this review is to elucidate the pathomechanism of SJS and TEN in genetic and immunologic point of views.
GENETIC MARKERS RELATED TO SJS! TEN Genetic association between HLA alleles and SJS! TEN was found from several reports, and the ethnicity specific feature is emphasized these years. A significantly strong correlation was first found in Han TEN patients Chinese in 2004.12 CBZ-induced SJS! carried 100% of HLA-B*1502 allele, and only 3% HLAB*1502 carriers tolerated CBZ.12 According to its absence in Caucasians13 and Japanese,14 HLA-B*1502 allele seems uniquely limited in Han Chinese ancestral Asians and might be an explanation for the extremely high risk of CBZ-induced SJS! TEN in Southeast Asians comparing to Caucasians and Japanese. Later studies conducted in the South-East countries, including Hong Kong, Malaysia, India, Singapore, Thailand, Vietnam, Indonesia, and Philippines, were further confirming its high prevalence (2.3% to 8.4%) of this ethnic specific allele.15 Because of the ethnic specialty in HLA-B*1502, CBZ and some similar structural anticonvulsants were fully studied. One hundred percent CBZ-induced SJS was HLA-B*1502 in Thailand people.16 Malay and Chinese carried 15.7% and 5.7% of HLA-B*1502, respectively, and had 75% incidence of HLA-B*1502 in CBZ-SJS or TEN.17 A lower prevalence (2.5%)18 and 75% incidence of HLA-B*1502 in CBZ-induced SJS were reported in Indians.19 Other than HLA-B*1502, HLA-B*5901 has been suggested as a candidate marker in CBZ-induced SJS in Japanese with 15.16 relative risk20; however, due to
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its small sample size (10 patients), further investigation is needed to validate this finding. In addition, HLA-A*0206, was proposed as a marker in SJS! TEN according to the ocular complications in Japanese.21 HLA-B*5801 allele is another genetic marker found to have an association with a commonly prescribed drug for decreasing uric acid level in hyperuricemia―allopurinol. In Taiwanese, it was 100% presented in patients with allopurinol-induced severe cutaneous adverse reactions, but only 15% in allopurinol tolerant patients.22 The same strong association was confirmed in a Thai population.23 However, findings from Japanese population remain to clarify. A moderate association (4 out of 10) in a Japanese population14 was reported. Opposite to this finding, Kano et al. suggested that HLA-B*4801 but not B*5801 was associated with CBZ-induced drug rash with eosinophilia and systemic symptoms (DRESS).24 In addition, the ethnic limitation also presents in HLA-B* 5801 on its low allele frequency in Caucasians.25 Moreover, an antiretroviral drug, abacavir, can cause drug hypersensitivity and was linked to HLA-B* 5701 allele. The combination of HLA-B*5701, HLADR7, and HLA-DQ3 once reported having a 100% predictability of the abacavir-induced hypersensitivity.26 Further study demonstrated that the prevalence of HLA-B*5701 was high in Caucasians and suggested that prior screening of the carriage of HLA-B*5701 could effectively reduce the abacavir hypersensitivity.27 In contrast to the high prevalence of HLA-B* 5701 and incidence of abacavir hypersensitivity, none of the patients with Asian ancestry in Korea carried HLA-B*5701.28
DRUG-SPECIFIC, HLA-DEPENDENT, T-CELLMEDIATED IMMUNITY IN SJS! TEN The discoveries of the involvement of HLAdependent presentation of an offending drug or its metabolites for T-cell activation29,30 demolished the “hapten hypothesis”31 in severe drug hypersensitivity. The proposed pharmoco-immune (p-i) concept32 is the mainstream explanation for the drug-induced delay cutaneous adverse reactions, suggesting the direct and non-covalent binding between a drug and Tcell receptors (TCR) with HLA molecules takes the responsibility for the drug-induced immunity (Fig. 1). The pathogenesis of the induction of cytotoxic responses in SJS! TEN is generated by the recognition of offending drugs to HLA class I molecule initiated T-cell activation which results a clonal expansion of CD8+ cytotoxic T-cells in skin.33 Our findings, the strong associations between HLA-B*1502 and CBZ12 as well as the HLA-B*5801 and allopurinol,22 support that drug-induced SJS! TEN is a HLA-restricted immunity.12 Furthermore, our later results verified 5 peptides showing high affinities for HLA-B*1502, providing CBZ recognition of HLA, locating at antigen presenting cells.34,35
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Genetics and Signals in SJS! TEN
CBZ
HLAB*1502
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CD8+ Cytotoxic T-cell TCR
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Fi g.1 A modelofker at i noc y t eapopt os i si nduc edbyt hei mmune synapseofdr ugHLATCR i nt er ac t i oni nSJ S/ TEN.Asi l l us t r at ed,t he i mmuner esponsemaybet r i gger edbyt hebi ndi ngofanant i geni c dr ug( e. g.car bamazepi ne[ CBZ] ) ,t oas pec i f i cHLAal l el e( e. g.HLAB* 1502)onaker at i noc y t e,whi c har et hemai nant i genpr es ent i ng cel l s( APC)i nSJS/ TEN.Then,s pec i f i cTc el lr ec ept or s( TCR)oft he CD8+ cyt ot oxi cT l ymphoc y t es ( CTLs )r ec ogni z et he dr ugHLA compl ex.Upont heact i v at i on,CTLsorNKTc el l spr oduc ec y t ok i nes andchemoki nes,aswel last hec y t ot ox i cpr ot ei ns ,par t i c ul ar l y ,s ec r et or ygr anul ysi nandl eadt oex t ens i v ek er at i noc y t eapopt os i s .
INVOLVEMENT OF NATURAL KILLER CELLS IN SJS! TEN In addition to CTLs, NK cells also involves in SJS! TEN. The blister cells in skin lesions of SJS! TENaffected patients were mainly CTLs and NK cells36; moreover, HLA class I-binding proteins have activating (KAR, killer activating receptor) or inhibitory (KIR, killer inhibitory receptor) cytolytic functions for regulating NK cells.37 Nassif A. et al.29 further demonstrated an observation of the infiltration of CTLs and NK cells in skin lesions in TEN patients. Our recent study also demonstrated that granulysin, secreted from CTLs and NK cells, is a key effector responsible for keratinocytes death in SJS! TEN.1
DANGER SIGNALS INDUCING EXTENSIVE KERATINOCYTE APOPTOSIS IN SJS! TEN Upon proposed immunopathogenesis of SJS! TEN, the general agreed central hypothesis is the T-cellmediated massive apoptosis in keratinocytes. Until recently, there are three reported pathways advocated as basic effectors: Fas-FasL interaction, perforin! granzyme B, and the granulysin.
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Fas-FasL-INDUCED APOPTOSIS Although Fas-FasL-induced apoptosis in keratinocytes is one of the most thoroughly studied immune mechanism in SJS! TEN, inconsistent findings of Fas, FasL, and soluble FasL (sFasL) questioned the original hypothesis from Viard et al.11 Fas was discovered to cause cell death upon binding with its ligand in SJS! TEN. Viard et al.11 suggested the activated Fas servers as a death receptor in triggering apoptosis of keratinocytes in SJS! TEN. The cytoplasmic death domain of Fas undergoes conformational changes upon recognition of FasL. The Fas-FasL then recruits a Fas-associated death domain protein (FADD) which has a affinity to bind to both of the Fas death domain and procaspase 8. Once the procaspase 8 is recruited by FADD, the multiple copies of procaspase 8 are brought together and autoactivate themselves to caspase 8 which triggers the caspase cascade for intracellular DNA degradation.38 One study concluded the same result as Viard et al. by incubating cell-free supernatants of blister fluid containing sFasL secreted from keratinocytes and not resulting in apoptosis in keratinocytes.39 Viard et al. also showed that FasL presented on the
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Chung WH et al. cell surface of keratinocytes in TEN patients and sFasL was found to have high levels in the serum, but not in patients with maculopapular drug reaction or normal persons.11 Metalloproteinases (MPs) present at cell surface in many tissue types, including keratinocytes40 and cleave FasL into sFasL at its TNFhomologous portion.41,42 The high sFasL serum level observed in TEN patients might cause by the MP cleavage reaction. A study also consistently found an apparent elevation of sFasL within 2 days after the onset of skin damage in a TEN patient.43 Testing the serological sFasL for a short period at the beginning of onset may help to define the progress of SJS! TEN. Controversial interpretations disagreed with either the source of FasL or its role in apoptosis effector. Studies demonstrated that FasL is not located at the extracellular membrane surface of keratinocytes, but rather transporting to the cell surface upon suffering keratinocyte damage.40,44 Abe et al.45 found that the apoptosis of cultured keratinocytes was induced by adding high levels of sFasL containing sera isolated from a SJS! TEN patient, and was blocked by addition of anti-FasL monoclonal antibody. They also showed the peripheral blood mononuclear cells (PBMCs) of TEN patients were the sites produced high levels of sFasL.45 Since Amato et al.46 first reported the treatment of intravenous immunoglobulins (IVIG) in a SJS patient, many studies subsequently applied IVIG to treat the SJS and TEN patients. IVIG therapy is a treatment based on the hypothesis of Fas-FasL interaction, by blocking FasL binding to the Fas receptor interferes with the downstream signaling for triggering apoptosis of the keratinocytes.11 Although evidence seemed to show the effectiveness of IVIG in in vitro studies,11,47 benefits were inconsistently seen in neither the mortality nor the progression of skin detachment,48-52 which yield the controversial role for the IVIG treatment in SJS! TEN.
PERFORIN! GRANZYME B APOPTIC PATHWAY High concentrations of granzyme B was found in TEN blister fluid.53 Nassif A. et al.54 presented a contradictory hypothesis to the Fas-FasL interaction by showing that the cytotoxicity from the blister fluid mononuyclear cells in TEN could be blocked by inhibitors of perforin! granyzyme B rather then blocked by an anti-Fas monoclonal antibody. Perforin and granzyme B are stored in secretory granules of activated CTLs and NK cells.55 Perforin binds and punches a channel in the membrane of target cells for entering of the granzyme B to activate the caspase cascade and its following apoptotic pathways.56,57
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OTHER CYTOKINES AND SIGNALS INVOLVED IN THE PATHOGENESIS OF SJS! TEN In addition to Fas-FasL or perforin! granzyme B pathways, some cytokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ,39 and interleukin (IL)-10,39 were also found to be up-regulated in SJS! TEN. Blister cells of SJS! TEN were reported to secret IFN-γ and stimulate keratinocytes to express TNF-α, FasL, and IL-10 which were present in higher concentrations in the blister fluids as a defense mechanism against CTLs.39 TNF-α has been reported abundantly presenting in the keratinocytes of epidermis,58,59 blister fluid mononuclear cells and macroTEN. TNF-α has phages53,58,59 and PBMCs60 in SJS! been suggested having an up-regulating function in Fas and FasL60 and via the activation of TNF-receptor 1 (TNF-R1), initiating the downstream FADD and caspases.61,62 Apoptosis activation in TEN patients had been implied to be induced by TNF.10 In addition, little evidence showed that TNF-α also can activate TNF-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-Rs), which may result in the activation of FADD apoptotic pathway.63 Interestingly, contradictory to TNF-α apoptosis inducing role, its binding to TNF-R1 can up-regulate NF-κB64 and associate with an anti-apoptotic in keratinocytes.65 Anti-TNF therapy once was thought to be a possible potential treatment for TEN patients; however, no beneficial effect was concluded from anti-TNF by thalidomide in TEN.66 There was one study that supported both of the Fas-FasL and perforin! granzyme B pathways in keratinocytes apoptosis.53 This study found an increasing pattern of TNF-α, perforin, granzyme B, and FasL accompanied with the severity, ranging from the mildest maculopapular rashes to the severest TEN, and suggested that the perforin! granzyme B played as the major effectors and Fas-FasL interaction was probably related to the severer adverse drug reactions.
GRANULYSIN IS THE MAJOR FACTOR FOR KERATINOCYTE APOPTOSIS IN SJS! TEN Recently, we found secretory granulysin servers as a major cytotoxic molecule responsible for widespread keratinocyte necrosis in SJS! TEN,1 instead of those previously reported sFasL, granzyme B, or perforin. We performed global gene expression profiling of the blister cells and found that granulysin RNA was the most significant cytotoxic molecule expressed. Granulysin protein concentrations in the SJS! TEN blister fluids were two to four orders of magnitude higher than perforin, granzyme B or sFasL concentrations, and depleting granulysin reduced the cytotoxicity (Fig. 2). Westernblot analysis showed that granulysin in the fluids was predominantly the 15-
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Genetics and Signals in SJS! TEN
Keratinocyte
CLT
Epidermis
NK/NKT Apoptotic keratinocyte
Blister
sFasL Perforin Grazyme B
Blood vessel
Fibroblast
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Fi g.2 Pat hogenesi sofepi der malnecr osi sanddi s s emi nat edk er at i noc y t eapopt os i si nSJ S/ TEN.Duet ot he si gnal sofi mmunesynapse,t heCTLsandNK/ NKTc el l si mmi gr at et ot heepi der mi sofs k i n.CTLs ,andNK/ NKT cel l spr oduceal ar geamountofi mmunemedi at or s( e. g. ,s ol ubl eFas L[ s Fas L] ,per f or i n,gr az y meB,andgr anul ysi n)i nt ot heext r acel l ul arspace.I npar t i cul ar ,s ec r et or ygr anul y s i n,ex pr es s edatav er yhi ghl ev eli nt hes k i nl esi ons,i samai nweaponofCTLs,NK,andNKTc el l s ,at t ac k sk er at i noc y t esandr es ul t si nex t ens i v eepi der mal necr osi sandbl i st erf or mat i on.Bycompar i son,gr anz y meB/ per f or i nandFas / Fas Lar epr oduc edv i agr anul eex ocyt osi suponcel l cel l cont act ,andpr esenti nl owerc onc ent r at i onst hangr anul y s i ni nt hei nf l ammat or yl es i ons .Af t erencount er i ng t he at t acksoft hese cyt ot ox i cpr ot ei ns ,k er at i noc y t esar e damaged and t hen t he c as pas e si gnal i ngpat hwayt ur nson,l eadi ngt oapopt os i spr ogr es s .
kDa secretory form. In vitro, purified 15-kDa granulysin exhibited significant cytotoxicity at the level presenting in the SJS! TEN blister fluids. However, sFasL, perforin, and granzyme B concentrations in the SJS! TEN blister fluid had minimum cytotoxicity. A further injection of granulysin into mouse skin resulted in a SJS-TEN-alike skin necrosis.1 Thus, our findings demonstrated that granulysin, not granzyme B, perforin or sFasL as previously implicated, is the key molecule responsible for the disseminated keratinocyte death in SJS! TEN. Following this line, Abe et al.45 further reported that an increasing serum level of granulysin could serve as an early diagnostic biomarker for SJS! TEN. Granulysin is a cationic cytolytic protein produced by CTLs, NK and NKT cells.67 The 15-kDa granulysin was proposed as a precursor of the 9-kDa form.68,69 In addition to having the cytotoxic effect, granulysin also was demonstrated its chemoattractant ability for T lymphocytes, monocytes and other inflammatory cells, and activation function in the expression of a number of cytokines, including RANTES! CCL5, MCP-1, MCP-3, MIP-1α! CCL3, IL-10, IL-1, IL-6 and
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IFN-α.70 However, much of SJS! TEN remains in mystery. How does taking a drug lead to secretion of granulysin? How does CD8+T! NK and NKT cells regulate the secretion of granulysin in SJS! TEN? The specific relationship among offending drugs, HLA alleles, and cytotoxic signals from CLTs! NK! NKT cells in SJS! TEN remains further investigation.
CONCLUSION Increasing data have revealed that the genetic predisposition and immune mediators play important roles in the drug hypersensitivity. As reviewed in this article, it is known that HLA alleles associated with SJS! TEN may be variable among different human population. These HLA alleles may be not only responsible for the genetic susceptibility, but also play a pathogenesis role, which may present the drugs! metabolites to CTLs for the initiation of the downstream danger signals in the disease. In addition to Fas-FasL, perforin! granzyme B biomarkers, the secretory 15 kDa granulysin, is now known to be the major weapon of CTLs! NK! NKT cells and leads to the ex-
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Chung WH et al. tensive epidermal necrolysis in SJS! TEN. Understanding the molecular mechanism of the interaction of HLA, offending drugs and TCR, as well as CTLs! NK cells activation, would facilitate the development of new approaches for the management of SJS! TEN. In conclusion, those transitional studies offer us a better understanding of the immune mechanisms, biomarkers for disease prevention and early diagnosis, as well as providing the therapeutic target for the treatment of SJS! TEN.
ACKNOWLEDGEMENTS This work was supported by grants from the National Science Council, Taiwan (NSC 98-2314-B-182A-027MY3, 98-2320-B-010-002-MY3), and grants from Chang-Gung Memorial Hospital (BMRPG-290011, CMRPG-290051).
REFERENCES 1. Chung WH, Hung SI, Yang JY et al. Granulysin is a key mediator for disseminated keratinocyte death in StevensJohnson syndrome and toxic epidermal necrolysis. Nat Med 2008;14:1343-50. 2. Roujeau JC. The spectrum of Stevens-Johnson syndrome and toxic epidermal necrolysis: a clinical classification. J Invest Dermatol 1994;102:28S-30. 3. Roujeau JC, Stern RS. Severe adverse cutaneous reactions to drugs. N Engl J Med 1994;331:1272-85. 4. Chan HL, Stern RS, Arndt KA et al. The incidence of erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis. A population-based study with particular reference to reactions caused by drugs among outpatients. Arch Dermatol 1990;126:43-7. 5. Di Pascuale MA, Espana EM, Liu DT et al. Correlation of corneal complications with eyelid cicatricial pathologies in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis syndrome. Ophthalmology 2005;112: 904-12. 6. Revuz J, Penso D, Roujeau JC et al. Toxic epidermal necrolysis. Clinical findings and prognosis factors in 87 patients. Arch Dermatol 1987;123:1160-5. 7. Lebargy F, Wolkenstein P, Gisselbrecht M et al. Pulmonary complications in toxic epidermal necrolysis: a prospective clinical study. Intensive Care Med 1997;23:123744. 8. Sugimoto Y, Mizutani H, Sato T, Kawamura N, Ohkouchi K, Shimizu M. Toxic epidermal necrolysis with severe gastrointestinal mucosal cell death: a patient who excreted long tubes of dead intestinal epithelium. J Dermatol 1998;25:533-8. 9. Ducic I, Shalom A, Rising W, Nagamoto K, Munster AM. Outcome of patients with toxic epidermal necrolysis syndrome revisited. Plast Reconstr Surg 2002;110:768-73. 10. Paul C, Wolkenstein P, Adle H et al. Apoptosis as a mechanism of keratinocyte death in toxic epidermal necrolysis. Br J Dermatol 1996;134:710-4. 11. Viard I, Wehrli P, Bullani R et al. Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. Science 1998;282:490-3. 12. Chung WH, Hung SI, Hong HS et al. Medical genetics: a marker for Stevens-Johnson syndrome. Nature 2004;428: 486. 13. Lonjou C, Thomas L, Borot N et al. A marker for Stevens-
330
Johnson syndrome...: ethnicity matters. Pharmacogenomics J 2006;6:265-8. 14. Kaniwa N, Saito Y, Aihara M et al. HLA-B locus in Japanese patients with anti-epileptics and allopurinol-related Stevens-Johnson syndrome and toxic epidermal necrolysis. Pharmacogenomics 2008;9:1617-22. 15. Allele Frequencies in Worldwide Populations. Available from http:! ! www.allelefrequencies.net! . 16. Locharernkul C, Loplumlert J, Limotai C et al. Carbamazepine and phenytoin induced Stevens-Johnson syndrome is associated with HLA-B*1502 allele in Thai population. Epilepsia 2008;49:2087-91. 17. Chang CC, Too CL, Murad S. Association of HLA-B*1502 with carbamazepine-induced toxic epidermal necrolysis and Stevens-Johnson Syndrome in Malaysian population. Proceeding in 7th Asian-Oceanian Epilepsy Congress, Xiamen. 2008. 18. Rajalingam R, Parham P, Mehra NK. HLA-A, -B, -Cw, -DQA1, -DQB1 and -DRB1 alleles and KIR alleles in a Hindu population from Punjab, India. Human Immunology 2004;65:958-60. 19. Mehta TY, Prajapati LM, Mittal B et al. Association of HLA-B*1502 allele and carbamazepine-induced StevensJohnson syndrome among Indians. Indian J Dermatol Venereol Leprol 2009;75:579-82. 20. Ikeda H, Takahashi Y, Yamazaki E et al. HLA class I markers in Japanese patients with carbamazepineinduced cutaneous adverse reactions. Epilepsia 2010;51: 297-300. 21. Ueta M, Tokunaga K, Sotozono C et al. HLA class I and II gene polymorphisms in Stevens-Johnson syndrome with ocular complications in Japanese. Mol Vis 2008;14:550-5. 22. Hung SI, Chung WH, Liou LB et al. HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol. Proc Natl Acad Sci U S A 2005; 102:4134-9. 23. Tassaneeyakul W, Jantararoungtong T, Chen P et al. Strong association between HLA-B*5801 and allopurinolinduced Stevens-Johnson syndrome and toxic epidermal necrolysis in a Thai population. Pharmacogenet Genomics 2009;19:704-9. 24. Kano Y, Hirahara K, Asano Y, Shiohara T. HLA-B allele associations with certain drugs are not confirmed in Japanese patients with severe cutaneous drug reactions. Acta Derm Venereol 2008;88:616-8. 25. Lonjou C, Borot N, Sekula P et al. A European study of HLA-B in Stevens-Johnson syndrome and toxic epidermal necrolysis related to five high-risk drugs. Pharmacogenet Genomics 2008;18:99-107. 26. Mallal S, Nolan D, Witt C et al. Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to HIV-1 reverse-transcriptase inhibitor abacavir. Lancet 2002;359:727-32. 27. Mallal S, Phillips E, Carosi G et al. HLA-B*5701 screening for hypersensitivity to abacavir. N Engl J Med 2008;358: 568-79. 28. Park WB, Choe PG, Song KH et al. Should HLA-B*5701 screening be performed in every ethnic group before starting abacavir? Clin Infect Dis 2009;48:365-7. 29. Nassif A, Bensussan A, Boumsell L et al. Toxic epidermal necrolysis: effector cells are drug-specific cytotoxic T cells. J Allergy Clin Immunol 2004;114:1209-15. 30. Schnyder B, Mauri-Hellweg D, Zanni M, Bettens F, Pichler WJ. Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alphabeta T cell clones. J Clin Invest 1997;100:136-41.
Allergology International Vol 59, No4, 2010 www.jsaweb.jp!
Genetics and Signals in SJS! TEN
31. Weltzien HU, Moulon C, Martin S, Padovan E, Hartmann
49. Schneck J, Fagot JP, Sekula P, Sassolas B, Roujeau JC,
U, Kohler J. T cell immune responses to haptens. Structural models for allergic and autoimmune reactions. Toxicology 1996;107:141-51. 32. Pichler WJ. Pharmacological interaction of drugs with antigen-specific immune receptors: the p-i concept. Curr Opin Allergy Clin Immunol 2002;2:301-5. 33. Naisbitt DJ, Britschgi M, Wong G et al. Hypersensitivity reactions to carbamazepine: characterization of the specificity, phenotype, and cytokine profile of drug-specific T cell clones. Mol Pharmacol 2003;63:732-41. 34. Yang CW, Hung SI, Juo CG et al. HLA-B*1502-bound peptides: implications for the pathogenesis of carbamazepineinduced Stevens-Johnson syndrome. J Allergy Clin Immunol 2007;120:870-7. 35. Barber LD, Percival L, Arnett KL, Gumperz JE, Chen L, Parham P. Polymorphism in the alpha 1 helix of the HLAB heavy chain can have an overriding influence on peptide-binding specificity. J Immunol 1997;158:1660-9. 36. David V, Bourge JF, Guglielmi P, Mathieu-Mahul D, Degos L, Bensussan A. Human T cell clones use a CD3associated surface antigen recognition structure to exhibit both NK-like and allogeneic cytotoxic reactivity. J Immunol 1987;138:2831-6. 37. Le Cleach L, Delaire S, Boumsell L et al. Blister fluid T lymphocytes during toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors. Clin Exp Immunol 2000;119: 225-30. 38. Janeway CA, Travers P, Walport M, Schlomchik M. Immunobiology: The Immune System in Health and Disease, 5th edn. New York: Garland Publishing, 2001. 39. Nassif A, Moslehi H, Le Gouvello S et al. Evaluation of the potential role of cytokines in toxic epidermal necrolysis. J Invest Dermatol 2004;123:850-5. 40. Viard-Leveugle I, Bullani RR, Meda P et al. Intracellular localization of keratinocyte Fas ligand explains lack of cytolytic activity under physiological conditions. J Biol Chem 2003;278:16183-8. 41. Tanaka M, Itai T, Adachi M, Nagata S. Downregulation of Fas ligand by shedding. Nat Med 1998;4:31-6. 42. Knox PG, Milner AE, Green NK, Eliopoulos AG, Young LS. Inhibition of metalloproteinase cleavage enhances the cytotoxicity of Fas ligand. J Immunol 2003;170:677-85. 43. Chang HY, Cooper ZA, Swetter SM, Marinkovich MP. Kinetics and specificity of fas ligand induction in toxic epidermal necrolysis. Arch Dermatol 2004;140:242-4. 44. Filipowicz E, Adegboyega P, Sanchez RL, Gatalica Z. Expression of CD95 (Fas) in sun-exposed human skin and cutaneous carcinomas. Cancer 2002;94:814-9. 45. Abe R, Shimizu T, Shibaki A, Nakamura H, Watanabe H, Shimizu H. Toxic epidermal necrolysis and StevensJohnson syndrome are induced by soluble Fas ligand. Am J Pathol 2003;162:1515-20. 46. Amato GM, Travia A, Ziino O. [The use of intravenous high-dose immunoglobulins (IGIV) in a case of StevensJohnson syndrome]. Pediatr Med Chir 1992;14:555-6 (in Italian). 47. Wehrli P, Viard I, Bullani R, Tschopp J, French LE. Death receptors in cutaneous biology and disease. J Invest Dermatol 2000;115:141-8. 48. Bachot N, Revuz J, Roujeau JC. Intravenous immunoglobulin treatment for Stevens-Johnson syndrome and toxic epidermal necrolysis: a prospective noncomparative study showing no benefit on mortality or progression. Arch Dermatol 2003;139:33-6.
Mockenhaupt M. Effects of treatments on the mortality of Stevens-Johnson syndrome and toxic epidermal necrolysis: A retrospective study on patients included in the prospective EuroSCAR Study. J Am Acad Dermatol 2008;58: 33-40. 50. Valeyrie-Allanore L, Wolkenstein P, Brochard L et al. Open trial of ciclosporin treatment for Stevens-Johnson syndrome and toxic epidermal necrolysis. Br J Dermatol. Epub 2010 May 25. 51. Wolf R, Davidovici B. Severe cutaneous adverse drug reactions: who should treat, where and how?: Facts and controversies. Clin Dermatol 2010;28:344-8. 52. Mittmann N, Chan B, Knowles S, Cosentino L, Shear N. Intravenous immunoglobulin use in patients with toxic epidermal necrolysis and Stevens-Johnson syndrome. Am J Clin Dermatol 2006;7:359-68. 53. Posadas SJ, Padial A, Torres MJ et al. Delayed reactions to drugs show levels of perforin, granzyme B, and Fas-L to be related to disease severity. J Allergy Clin Immunol 2002;109:155-61. 54. Nassif A, Bensussan A, Dorothee G et al. Drug specific cytotoxic T-cells in the skin lesions of a patient with toxic epidermal necrolysis. J Invest Dermatol 2002;118:728-33. 55. Tschopp J, Nabholz M. Perforin-mediated target cell lysis by cytolytic T lymphocytes. Annu Rev Immunol 1990;8: 279-302. 56. Lowin B, Peitsch MC, Tschopp J. Perforin and granzymes: crucial effector molecules in cytolytic T lymphocyte and natural killer cell-mediated cytotoxicity. Curr Top Microbiol Immunol 1995;198:1-24. 57. Darmon AJ, Nicholson DW, Bleackley RC. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature 1995;377:446-8. 58. Paquet P, Nikkels A, Arrese JE, Vanderkelen A, Pierard GE. Macrophages and tumor necrosis factor alpha in toxic epidermal necrolysis. Arch Dermatol 1994;130:6058. 59. Paquet P, Pierard GE. Erythema multiforme and toxic epidermal necrolysis: a comparative study. Am J Dermatopathol 1997;19:127-32. 60. Arnold R, Seifert M, Asadullah K, Volk HD. Crosstalk between keratinocytes and T lymphocytes via Fas! Fas ligand interaction: modulation by cytokines. J Immunol 1999;162:7140-7. 61. Zhuang L, Wang B, Shinder GA, Shivji GM, Mak TW, Sauder DN. TNF receptor p55 plays a pivotal role in murine keratinocyte apoptosis induced by ultraviolet B irradiation. J Immunol 1999;162:1440-7. 62. Ruckert R, Lindner G, Bulfone-Paus S, Paus R. High-dose proinflammatory cytokines induce apoptosis of hair bulb keratinocytes in vivo. Br J Dermatol 2000;143:1036-9. 63. Leverkus M, Neumann M, Mengling T et al. Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes. Cancer Res 2000;60:553-9. 64. Wang CY, Cusack JC Jr, Liu R, Baldwin AS Jr. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaB. Nat Med 1999;5:412-7. 65. Qin JZ, Chaturvedi V, Denning MF, Choubey D, Diaz MO, Nickoloff BJ. Role of NF-kappaB in the apoptoticresistant phenotype of keratinocytes. J Biol Chem 1999; 274:37957-64. 66. Wolkenstein P, Latarjet J, Roujeau JC et al. Randomised comparison of thalidomide versus placebo in toxic epider-
Allergology International Vol 59, No4, 2010 www.jsaweb.jp!
331
Chung WH et al.
mal necrolysis. Lancet 1998;352:1586-9.
67. Gamen S, Hanson DA, Kaspar A, Naval J, Krensky AM, Anel A. Granulysin-induced apoptosis. I. Involvement of at least two distinct pathways. J Immunol 1998;161:1758-64. 68. Stenger S, Hanson DA, Teitelbaum R et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998;282:121-5. 69. Hanson DA, Kaspar AA, Poulain FR, Krensky AM. Bio-
332
synthesis of granulysin, a novel cytolytic molecule. Mol Immunol 1999;36:413-22. 70. Deng A, Chen S, Li Q, Lyu SC, Clayberger C, Krensky AM. Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator. J Immunol 2005; 174:5243-8.
Allergology International Vol 59, No4, 2010 www.jsaweb.jp!